| With the intensification of eutrophication,the content of ammonia in water will increase.Therefore,it is necessary to explore the influence of ammonia on B.fatalis,which is one of the dominant species in eutrophic water.In this study,we carried out acute toxicity test,chronic toxicity test and transcriptome sequencing analysis,to explore the effects of ammonia on the growth performance,internal morphological changes and antioxidant related enzyme activities of B.fatalis in different ammonia concentration and reveal its internal molecular mechanism.The major findings of this study are as follows:(1)The LC50 values of ammonia on B.fatalis and the effects of different ammonia concentrations on the growth performance of B.fatalis were determined.The results indicated that the 24 h LC50,48 h LC50,72 h LC50,and 96 h LC50 values of NH3 on B.fatalis were 2 0.59,13.06,5.77,and 3.36 mg·L-1,respectively.We set the ammonia concentration as four gradients of 0,0.22,0.57 and 1.4 mg·L-1 in the chronic toxicity experiment.The 7-days NH3 exposure results showed that NH3 at low and medium doses(0.22 and 0.57 mg·L-1)displayed stimulative effects on the B.fatalis population and the body length of the newborns(0.22 mg·L-1 NH3,p<0.05,0.57 mg·L-1 NH3,p<0.01).The 20-days NH3 exposure results showed that the body length of B.fatalis at the first reproduction in control group(0.320 mm)was lower than that of B.fatalis in ammonia concentration of 0.22 mg·L-1(0.356 mm),and 0.57 mg·L-1(0.390 mm)(P<0.05),but that of B.fatalis in ammonia concentration of 1.4 mg·L-1(0.320 mm)was not significantly different from that of control group;and the body length and the first reproduction time of B.fatalis in ammonia concentration subjects was no significant difference.(2)Relevant antioxidant enzyme activity indicators(ACP,SOD,CAT,GSH-Px,MDA)were measured for B.fatalis under ammonia stress,the results showed that the antioxidant stress response of B.fatalis was induced by different ammonia concentrations at 6 h and 12 h,but was inhibited after 24 h.And the effect of ammonia on the internal structure of B.fatalis was studied by transmission electron microscope(TEM).The results showed that significant increase in number of ribosomes at low doses,while the interior structure showed no marked change at high dose.The transcriptome analysis and RT-qPCR results showed that under ammonia stress,a large number of differential genes were enriched in ribosomal,Rapl signal,tuberculosis,phagosome,oxytocin signal and oxidative phosphorylation,among which the enrichment of ribosomal pathway was the most significant,indicating that under ammonia stress,Daphnia fragilis mainly responded to the high ammonia environment by regulating ribosomal protein,oxytocin signal and improving antioxidant capacity.The results of fluorescence quantitative detection showed that the relative expression levels of differential genes related to ribosomal protein structure(RPL10,RPL12,RPL27a,RPL10a and RPL19)and antioxidant stress(SOD,CAT,GSH-Px)were rapidly down regulated,which was consistent with the results of transcriptome analysis.(3)The transcriptome analysis results showed that a large number of differential genes were enriched in ribosomal,rapl signaling,tuberculosis,phagosome,oxytocin signaling and oxidative phosphorylation pathway under ammonia stress.The RT-qPCR results showed that relative expression levels of different genes related to ribosome structure(RPL10,RPL12,RPL27A,RPL10A and RPL19)and antioxidant stress(SOD,CAT and GSH-Px)were rapidly down-regulated under NH3 exposure,which was consistent with the results of transcriptome analysis.In a word,it is inferred that ammonia at a certain dose(NH3 ? 0.57 mg·L-1)stimulates the growth and reproduction of B.fatalis,the positive promoting effect is achieved by stimulating the number and function of ribosomes and enhancing the antioxidant stress ability of B.fatalis,which may be its defensive reaction to ammonia,especially in eutrophic water. |
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